Catalog Number AP 095
Intended Use
The QualiPlate Kit for Pepper Mild Mottle Virus screens for the presence of
Pepper Mild Mottle Tobamovirus (PMMV) in seed or leaf extracts. In studies
on seed lots determined to be PMMV positive by other test methods and by
comparison with controls, this kit was able to consistently detect the presence
of the virus (using minimum sample sizes of 3,000 seeds and minimum subsample
sizes of 500 seeds).
Preparation of Solutions
Wash Buffer: Add the contents of the packet of Wash Buffer Salts
(phosphate buffered saline, pH 7.4 – 0.05% Tween 20) to 1 liter of distilled
or deionized water, and stir to dissolve. Store refrigerated when not in use;
warm to room temperature* prior to assay. Additional 1L dry packets may
be purchased from Sigma Chemicals, Cat#P-3563, or similar recipes may
be prepared from salts on site.
1X Seed Extraction Buffer: Bring 10X Seed Extraction Buffer to room
temperature*, then stir or shake to dissolve precipitates completely before
proceeding. To make 1X Seed Extraction Buffer, add the entire 50 mL
bottle of 10X Seed Extraction Buffer to 450 mL of distilled or deionized
water in a suitable container, and mix thoroughly to dissolve any remaining
precipitates. Store 1X Seed Extraction Buffer refrigerated when not in use;
warm to room temperature* prior to assay. Additional 10X or 35X buffer
may be purchased from EnviroLogix (Cat#KR160 or Cat#KR186
respectively). See "Notes" section for preparing various volumes of Buffer.
*Please note: "room temperature" notation in all instructions is 18-
25°C – do not expose kit components or solutions to temperatures
above 25°C.
Sample Preparation
Seeds: The sample must be extracted with prepared 1X Seed Extraction
Buffer at a ratio of 1:10 (gram of seeds to mL of buffer). For example:
- 2.5 g of seed : 25 mL of 1X Seed Extraction Buffer
- 0.25 g of seed : 2.5 mL of 1X Seed Extraction Buffer
All seeds must be thoroughly ground/cracked in order for the internal tissue
to come in contact with the buffer. Use equipment appropriate for the size
of seed being tested (e.g. Polytron for most complete extraction, coffee
grinder, rubber mallet with mesh extraction bag). Soak ground seed tissue
in 1X Seed Extraction Buffer for 1 hour minimum at 4°C. Solids will settle
to the bottom; use the light-colored upper layer in the assay.
Leaf: The sample must be extracted with 1X Leaf Extraction Buffer at a ratio
of 1:10 (gram of leaf tissue to mL of buffer). For example:
- 0.1 g of leaf : 1 mL of 1X Leaf Extraction Buffer
All leaf tissue must be thoroughly macerated in order for ideal sample
extraction (e.g. EnviroLogix ACC 002 tube and pestle, mesh extraction
bags, bead-beater apparatus). Note: extracts will be foamy.
QualiPlate Kit for PMMV
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Rev. 07-22-11
Prepare Wash Buffer (and
Seed Extraction Buffer, if testing seed)
Remove unneeded strips
Add Extraction Buffer, controls,
and sample extracts
Mix plate, incubate
All incubation steps must be
performed on an orbital shaker
with 18+ mm orbital diameter
Pull off particle-free extract to run in the test. Clarification of extracts by
centrifugation is recommended (10 minutes at 1800-5000 x g), but not
required.
How to Run the Assay
Read all of these instructions before running the kit.
Allow all reagents to reach room temperature before beginning (at least 30
minutes with un-boxed plates and reagents at room temperature
(18-25°C) - do not remove strips from bag with desiccant until they have
warmed up).
Organize all reagents, sample extracts, and pipettes so that step 1 can
be performed in 15 minutes or less; the use of a multi-channel pipette is
strongly recommended for all reagent and extract transfers.
If more than four strips are to be run at one time, the 15 minutes is likely to
be exceeded, and the use of a multi-channel pipette is recommended (see
“Note” below).
If four or fewer strips are to be run, use a disposable-tip air-displacement
pipette and a clean pipette tip to add Extraction Buffer or sample extract to
the wells. Conjugate, Substrate, and Stop Solution may be added in the
same manner; alternatively, use a repeating pipette with a disposable tip on
the end of the Combitip for each of the three reagents.
If fewer than all twelve strips are used, reseal the unneeded strips and the
desiccant in the foil bag provided, and refrigerate.
Use the well identification markings on the plate edge as a guide when
adding the samples and reagents. It is recommended that at least two wells
each of 1X Seed or Leaf Extraction Buffer and known-negative seed or
leaf extract be run on each plate. Additional quality control samples may
be added at the discretion of the user. Sample extracts may be run in either
single or duplicate wells.
1. Add 100 µL of 1X Extraction Buffer, 100 µL of any user-prepared
negative control extract, and 100 µL of each sample extract to their
respective wells. Follow the same order of addition for all reagents. Treat
each plate as an independently timed assay.
NOTE: It is strongly recommended that a multi-channel pipette be used in
steps 1, 5, 8 and 9.
2. Thoroughly mix the contents of the wells by moving the plate in a rapid
circular motion on the bench top for 4-5 seconds. Be careful not to spill the
contents!
3. Cover the wells with tape or Parafilm to prevent evaporation and incubate
for 30 minutes at ambient temperature on an orbital shaker (with 18+
mm orbital diameter) at 150 to 200 rpm. Note: Shaking during
incubation steps is mandatory where called for. Failure to do so will result
in up to 50% loss in assay sensitivity.
Protocol option: For testing convenience, at this point samples may be
incubated overnight in the refrigerator (up to 16 hours at 5°C). Allow
plates to come to room temperature with the rest of the kit reagents the next
morning, before going on to step 4.
4. After incubation, carefully remove the covering and empty the contents of
the wells into a sink or other suitable container by inverting quickly and
vigorously shaking the plate. Flood the wells completely with Wash
Buffer, then empty as directed above. Repeat this wash step three more
QualiPlate Kit for PMMV
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Rev. 07-22-11
Bottle Wash method
Strip Plate Wash option
Add conjugate, mix, incubate, wash
Add substrate, mix, incubate
Add Stop Solution
Read plates in a Plate Reader
at 450 nm within 30 minutes of the
addition of Stop Solution.
times. After the final wash, keep the plate inverted and tap firmly on a dry
paper towel to remove as much Wash Buffer as possible.
If samples were incubated overnight, increase number of wash cycles to 8.
5. Add 100 µL of PMMV Enzyme Conjugate to each well.
6. Thoroughly mix the contents of the wells, as in step 2. Cover the wells
with new tape or Parafilm and incubate for 1 hour at ambient
temperature on an orbital plate shaker as described above. Note:
Shaking during incubation steps is mandatory where called for. Failure to
do so will result in up to 50% loss in assay sensitivity.
7. Wash the wells again as described in step 4. Alternatively, perform four
washes (300 µL/well) with a microtiter plate or strip washer.
8. Add 100 µL of Substrate to each well. Thoroughly mix the contents of the
wells by moving the plate in a rapid circular motion on the bench top for
20-30 seconds. Cover the wells with new tape or Parafilm and incubate for
30 minutes (for best results) at ambient temperature.
9. Add 100 µL of Stop Solution to each well and mix briefly. This will
change the blue color in the wells to yellow. Read the plate at 450 nm,
with a reference wavelength between 600 and 650 nm. Read the stopped
plate within 30 minutes; color may fade beyond that time.
NOTE: Stop Solution is 1 N HCl. Handle carefully.
How to Interpret the Results
Spectrophotometric Measurement
Set the wavelength of the microtiter plate reader to 450 nanometers (nm). (If it
has dual wavelength capability, use 600, 630 or 650 nm as the reference
wavelength.)
Interpreting Results
Compare the Optical Density (OD) of the sample extracts to those of the mean
Extraction Buffer wells, or preferably, to known-negative seed or leaf extract
wells, to determine presence or absence of PMMV in the sample extract.
Samples with absorbances significantly greater than those of the Seed or Leaf
Extraction Buffer and/or negative extract wells are presumed to be positive for
PMMV.
General Guidelines:
Mean OD of Extraction Buffer wells should not exceed 0.10.
Mean OD of PMMV-free seed or leaf extracts should not exceed 0.15.
If test results consistently fall outside these guidelines, please contact
EnviroLogix’ technical service.
Precautions and Notes
Observe any applicable regulations, federal or state guidelines, or in-house
lab safety protocols when disposing of samples and kit reagents.
Store all QualiPlate components at 4°C to 8°C (39°F to 46°F) when not in
use.
Do not expose QualiPlate components to temperatures greater than 37°C
(99°F) or less than 2°C (36°F) for optimum performance.
Allow all reagents to reach ambient temperature (18-25°C) before use.
QualiPlate Kit for PMMV
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Rev. 07-22-11
Do not use kit components after the expiration date.
Do not use reagents or test plates from one QualiPlate with reagents or test
plates from a different QualiPlate type or different lot number.
Do not use samples prepared for analysis in other test kits; do not run
sample extracts prepared for this assay in other brands of test kits.
Do not expose Substrate to sunlight during pipetting or while incubating
in the test wells.
Be sure to read the results of stopped color development at 450 nm,
not 405 nm.
Do not dilute or adulterate test reagents or use samples not called for in the
test procedure.
Quality of results is dependent upon following the assay protocol as
directed.
As with all tests, it is recommended that results be confirmed by an
alternate method when necessary.
Preparing 1X Seed Extraction Buffer: The following table shows the
formulas for preparing alternative volumes of Seed Buffer. Always make
sure the concentrated buffer is in solution before using it.
10X Extraction Buffer
(KR160, 50 or 1000 mL)
Finished Volume
10L 5L 2L 0.5L
Start with water (L) 9 4.5 1.8 0.45
Add 10X Extraction
Buffer (mL)
1000
(1 lg bottle) 500 200 50
(1 sm bottle)
Follow steps in order when diluting 35X Seed Extraction Buffer:
35X Extraction Buffer
(KR186, 500 mL)
Finished Volume
35L 20L 17.5L 10L 5L 2L
1. Start with water (L) 34 19.43 17 9.71 4.86 1.94
2. Add PVP (g),
stir to dissolve 700 400 350 200 100 40
3. Add 35X Extraction
Buffer (mL)
1000 571 500
(1 bottle) 286 143 57
QualiPlate Kit for PMMV
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Rev. 07-22-11
For Technical Support
Contact Us At:
EnviroLogix
500 Riverside Industrial
Parkway
Portland, ME 04103-1486
USA
Tel: (207) 797-0300
Toll Free: 866-408-4597
Fax: (207) 797-7533
e-mail:
horticulture@envirologix.com
website:
www.envirologix.com
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